Directed evolution

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Not to be confused with Directed_evolution_(transhumanism).
An example of a possible round to evolve a protein-based fluorescent sensor for a specific analyte using two consecutive FACS sortings [clarification needed]

Directed evolution (DE) is a method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids toward a user-defined goal.[1]

A typical procedure[edit]

A typical directed-evolution experiment involves three steps:[citation needed]

  1. Diversification: The gene encoding the protein of interest is mutated and/or recombined at random to create a large library of gene variants. Techniques commonly used in this step are PCR and DNA shuffling.
  2. Selection: The library is tested for the presence of mutants (variants) possessing the desired property using a screen or selection. Screens enable the researcher to identify and isolate high-performing mutants by hand, while selections automatically eliminate all nonfunctional mutants.
  3. Amplification: The variants identified in the selection or screen are replicated manifold by PCR, enabling researchers to sequence their DNA in order to understand what mutations have occurred.

Together, these three steps are termed a "round" of directed evolution. Most experiments involve more than one round. In these experiments, the best products of the previous round are diversified in the next round to create a new library. At the end of the procedure, all evolved protein or RNA mutants are characterized using biochemical methods

Likelihood of success[edit]

The likelihood of success in a directed evolution experiment is directly related to the total library size, as evaluating more mutants increases the chances of finding one with the desired properties.[citation needed] Performing multiple rounds of evolution is useful not only because a new library of mutants is created in each round but also because each new library uses better mutants as templates. The experiment is analogous to climbing a hill on a landscape where elevation is a function of the desired property. The goal is to reach the summit, which represents the best mutant. Each round of selection samples mutants on all sides of the starting template and selects the mutant with the highest elevation, thereby climbing the hill. A new round samples mutants on all sides of this new template and picks the highest of these, and so on until the summit is reached.

In vivo and in vitro[edit]

Directed evolution can be performed in living cells (in vivo evolution) or may not involve cells at all (in vitro evolution). In vivo evolution has the advantage of selecting for properties in a cellular environment, which is useful when the evolved protein or RNA is to be used in living organisms, but in vitro evolution is often more versatile in the types of selections that can be performed. Furthermore, in vitro evolution experiments can generate larger libraries because the library DNA need not be inserted into cells, the currently limiting step.

Advantages[edit]

The advantage of the directed evolution approach is that the researcher need not understand the mechanism of the desired activity in order to improve it.[citation needed] An alternative method is rational design of site-directed mutagenesis based on X-ray crystallography data.

Uses[edit]

Directed evolution is frequently used for protein engineering as an alternative to rational design[2], but can also be used to investigate fundamental questions of enzyme evolution.[3]

Protein engineering[edit]

As a protein engineering tool, DE has been most successful in three areas:

  1. Improving protein stability for biotechnological use at high temperatures or in harsh solvents.[4][5]
  2. Improving binding affinity of therapeutic antibodies (Affinity maturation)[6] and the activity of de novo designed enzymes[7].
  3. Altering substrate specificity of existing enzymes,[8][9][10][11] often for use in industry).[12]

Evolution studies[edit]

The study of natural evolution is traditionally based on extant organisms and their genes. However, research is fundamentally limited by the lack of fossils (and particularly the lack of ancient DNA sequences)[13][14] and incomplete knowledge of ancient environmental conditions. DE investigates evolution in a controlled system of genes for individual enzymes[15][16][17], ribozymes[18] and replicators[19][20] (similar to experimental evolution of eukaryotes,[21][22] prokaryotes[23] and viruses[24]).

DE allows control of selection pressure, mutation rate and environment (both the abiotic environment such as temperature, and the biotic environment, such as other genes in the organism). Additionally, there is a complete record of all evolutionary intermediate genes. This allows for detailed measurements of evolutionary processes, for example epistasis, evolvability, adaptive constraint[25] fitness landscapes[26], and neutral networks[27].

See also[edit]

References[edit]

  1. ^ Stephen Lutz, Beyond directed evolution - semi-rational protein engineering and design, Curr Opin Biotechnol. 2010 December ; 21(6): 734–743.
  2. ^ Turner, NJ (August 2009). "Directed evolution drives the next generation of biocatalysts.". Nature chemical biology 5 (8): 567–73. PMID 19620998. 
  3. ^ Romero, PA; Arnold, FH (December 2009). "Exploring protein fitness landscapes by directed evolution.". Nature reviews. Molecular cell biology 10 (12): 866–76. PMID 19935669. 
  4. ^ Gatti-Lafranconi, P; Natalello, A; Rehm, S; Doglia, SM; Pleiss, J; Lotti, M (8 January 2010). "Evolution of stability in a cold-active enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation.". Journal of molecular biology 395 (1): 155–66. PMID 19850050. 
  5. ^ Zhao, H; Arnold, FH (January 1999). "Directed evolution converts subtilisin E into a functional equivalent of thermitase.". Protein engineering 12 (1): 47–53. PMID 10065710. 
  6. ^ Hawkins, RE; Russell, SJ; Winter, G (5 August 1992). "Selection of phage antibodies by binding affinity. Mimicking affinity maturation.". Journal of molecular biology 226 (3): 889–96. PMID 1507232. 
  7. ^ Giger, L; Caner, S; Obexer, R; Kast, P; Baker, D; Ban, N; Hilvert, D (August 2013). "Evolution of a designed retro-aldolase leads to complete active site remodeling.". Nature chemical biology 9 (8): 494–8. PMID 23748672. 
  8. ^ Shaikh, FA; Withers, SG (April 2008). "Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis.". Biochemistry and cell biology = Biochimie et biologie cellulaire 86 (2): 169–77. PMID 18443630. 
  9. ^ Cheriyan, M; Walters, MJ; Kang, BD; Anzaldi, LL; Toone, EJ; Fierke, CA (1 November 2011). "Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate.". Bioorganic & medicinal chemistry 19 (21): 6447–53. PMID 21944547. 
  10. ^ MacBeath, G; Kast, P; Hilvert, D (20 March 1998). "Redesigning enzyme topology by directed evolution.". Science (New York, N.Y.) 279 (5358): 1958–61. PMID 9506949. 
  11. ^ Toscano, MD; Woycechowsky, KJ; Hilvert, D (2007). "Minimalist active-site redesign: teaching old enzymes new tricks.". Angewandte Chemie (International ed. in English) 46 (18): 3212–36. PMID 17450624. 
  12. ^ Turner, NJ (August 2009). "Directed evolution drives the next generation of biocatalysts.". Nature chemical biology 5 (8): 567–73. PMID 19620998. 
  13. ^ Pääbo, S; Poinar, H; Serre, D; Jaenicke-Despres, V; Hebler, J; Rohland, N; Kuch, M; Krause, J; Vigilant, L; Hofreiter, M (2004). "Genetic analyses from ancient DNA.". Annual review of genetics 38: 645–79. PMID 15568989. 
  14. ^ Höss, M; Jaruga, P; Zastawny, TH; Dizdaroglu, M; Pääbo, S (1 April 1996). "DNA damage and DNA sequence retrieval from ancient tissues.". Nucleic acids research 24 (7): 1304–7. PMID 8614634. 
  15. ^ Bloom, JD; Arnold, FH (16 June 2009). "In the light of directed evolution: pathways of adaptive protein evolution.". Proceedings of the National Academy of Sciences of the United States of America. 106 Suppl 1: 9995–10000. PMID 19528653. 
  16. ^ Moses, AM; Davidson, AR (17 May 2011). "In vitro evolution goes deep.". Proceedings of the National Academy of Sciences of the United States of America 108 (20): 8071–2. PMID 21551096. 
  17. ^ Goldsmith, M; Tawfik, DS (August 2012). "Directed enzyme evolution: beyond the low-hanging fruit.". Current opinion in structural biology 22 (4): 406–12. PMID 22579412. 
  18. ^ Salehi-Ashtiani, K; Szostak, JW (1 November 2001). "In vitro evolution suggests multiple origins for the hammerhead ribozyme.". Nature 414 (6859): 82–4. PMID 11689947. 
  19. ^ Sumper, M; Luce, R (January 1975). "Evidence for de novo production of self-replicating and environmentally adapted RNA structures by bacteriophage Qbeta replicase.". Proceedings of the National Academy of Sciences of the United States of America 72 (1): 162–6. PMID 1054493. 
  20. ^ Mills, DR; Peterson, RL; Spiegelman, S (July 1967). "An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule.". Proceedings of the National Academy of Sciences of the United States of America 58 (1): 217–24. PMID 5231602. 
  21. ^ Marden, JH; Wolf, MR; Weber, KE (November 1997). "Aerial performance of Drosophila melanogaster from populations selected for upwind flight ability.". The Journal of experimental biology 200 (Pt 21): 2747–55. PMID 9418031. 
  22. ^ Ratcliff, WC; Denison, RF; Borrello, M; Travisano, M (31 January 2012). "Experimental evolution of multicellularity.". Proceedings of the National Academy of Sciences of the United States of America 109 (5): 1595–600. PMID 22307617. 
  23. ^ Barrick, JE; Yu, DS; Yoon, SH; Jeong, H; Oh, TK; Schneider, D; Lenski, RE; Kim, JF (29 October 2009). "Genome evolution and adaptation in a long-term experiment with Escherichia coli.". Nature 461 (7268): 1243–7. PMID 19838166. 
  24. ^ Heineman, RH; Molineux, IJ; Bull, JJ (August 2005). "Evolutionary robustness of an optimal phenotype: re-evolution of lysis in a bacteriophage deleted for its lysin gene.". Journal of molecular evolution 61 (2): 181–91. PMID 16096681. 
  25. ^ Arnold, FH; Wintrode, PL; Miyazaki, K; Gershenson, A (February 2001). "How enzymes adapt: lessons from directed evolution.". Trends in biochemical sciences 26 (2): 100–6. PMID 11166567. 
  26. ^ Aita, T; Hamamatsu, N; Nomiya, Y; Uchiyama, H; Shibanaka, Y; Husimi, Y (5 July 2002). "Surveying a local fitness landscape of a protein with epistatic sites for the study of directed evolution.". Biopolymers 64 (2): 95–105. PMID 11979520. 
  27. ^ Bloom, JD; Raval, A; Wilke, CO (January 2007). "Thermodynamics of neutral protein evolution.". Genetics 175 (1): 255–66. PMID 17110496. 

Further reading[edit]

External links[edit]